The overall objectives of this program project are to: a) broaden our understanding of the regulation of cholesterol and bile acid metabolism in the liver and in the intestines; b) define the pathway and enzymes involved in the formation of secondary bile acids by intestinal bacteria; and c) determine the mechanism by which hydrophobic bile salts cause cell injury and explore the basis for hepatoprotective action of ursodeoxycholate. The specific aims of this proposal are: 1)purification, characterization, and cloning of key enzymes in the bile acid biosynthetic pathway (cholesterol 7alpha-hydroxylase (C7alphaH), 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) and 12alpha- hydroxylase (12alphaH); 2) determination of molecular basis by which bile acids, cholesterol and selected hormones regulate C7alphaH; 3) to study the regulation of 3beta-HSD and 12alphaH; 4)determine the mechanism of regulation of HMG-CoA reductase (HMG-CoA-R) by bile acids; 5) purification, characterization and cloning of hepatic cholesteryl ester hydrolase (CEH); 6) to study the regulation of CEH by bile acids, cholesterol and other regulatory agents in conjunction with other enzymes involved in the maintenance of hepatic cholesterol homeostasis; 7) purification, characterization and cloning of enzymes responsible for bacterial transformation of primary to secondary bile acids; 8) cloning and sequencing of bacterial bile acid transport (binding) protein from selected bacteria; 9) elucidation of the mechanism by which hydrophobic bile acids cause hepatic injury, and by which ursodeoxycholic acid, a representative hydrophilic bile salt protects membranes from toxic bile salts; 10) to determine the effects of different bile salts alone or in combination with UDCA, on the membrane fluidity gradient employing fluoroscopic probes to assess anisotropy at different depths within the membranes; and 11) to determine the mechanism by which hydrophobic bile salts affect cholesterol and phospholipid transfer.